Lactobionic Acid BP Grade

Ph Eur
C12H22O12 (acid form) -- 358.3 -- 96-82-2
C12H20O11 (d-lactone) -- 340.3 -- 5965-65-1

DEFINITION
Mixture in variable proportions of 4-O-b-D-galactopyranosyl-D-gluconic acid and 4-O-b-Dgalactopyranosyl-D-glucono-1,5-lactone.
Content: 98.0 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS
Appearance: White or almost white powder.
Solubility: Freely soluble in water, slightly soluble in glacial acetic acid, in anhydrous ethanol and in methanol.
mp: About 125C with decomposition.

IDENTIFICATION
A. Infrared absorption spectrophotometry.
Comparisonılactobionic acid CRS.
If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in water, dry at 105C and record new spectra using the residues.
B. Thin-layer chromatography.
Test solution: Dissolve 10 mg of the substance to be examined in water and dilute to 1 ml with the same solvent.
Reference solution: Dissolve 10 mg of lactobionic acid CRS in water and dilute to 1 ml with the same solvent.
Plate TLC silica gel plate.
Mobile phase concentrated ammonia, ethyl acetate, water, methanol.
Application: 5 μl.
Development: Over 3/4 of the plate.
Detection: Spray 3 times with ammonium molybdate solution and heat in an oven at 110C for 15 min.
Results: The principal spot in the chromatogram obtained with the test solution is similar in position and colour to the principal spot in the chromatogram obtained with the reference solution.

TESTS
Appearance of solution: The solution is clear and not more intensely coloured than reference.
Specific optical rotation: + 23.0 to + 29.0 (anhydrous substance).
Dissolve 1.0 g in 80 ml of water and dilute to 100.0 ml with the same solvent. Allow to stand for 24 h.
Reducing sugars: Maximum 0.2 per cent, calculated as glucose.
Dissolve 5.0 g in 25 ml of water  with the aid of gentle heat. Cool and add 20 ml of cupricitric solution and a few glass beads. Heat so that boiling begins after 4 min and maintain boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per cent V/V solution of glacial acetic acid and 20.0 ml of 0.025 M iodine. With continuous shaking, add 25 ml of a mixture of 6 volumes of hydrochloric acid and 94 volumes of water and, when the precipitate has dissolved, titrate the excess of iodine with 0.05 M sodium thiosulphate using 1 ml of starch solution, added towards the end of the titration, as indicator. Not less than 12.8 ml of 0.05 M sodium thiosulphate is required.
Heavy metals: Maximum 20 ppm.
Water: Maximum 5.0 per cent, determined on 0.50 g.
Total ash: Maximum 0.2 per cent.

Lactobionic Acid USP NF Grade

C12H22O12 (acid form) --- 358.30 --- 96-82-2.
C12H20O11 (δ-lactone) 340.28       [5965-65-1].
4-O-β-Galactopyranosyl-d-gluconic acid.    
UNII: 65R938S4DV    
DEFINITION
Lactobionic Acid is a mixture in variable proportions of 4-O-β-d-galactopyranosyl-d-gluconic acid and 4-O-β-d-galactopyranosyl-d-glucono-1,5-lactone. It contains 98% toT 102%, on the anhydrous basis.
IDENTIFICATION
A. Infrared Absorption: [Note— If the spectra obtained show differences, dissolve the test substance and USP Lactobionic Acid RS separately in water, dry at 105C, and record new spectra using the residues. ]
B. Thin-Layer Chromatography: Acceptance criteria: The principal spot from the Sample solution is similar in position and color to the principal spot from the Standard solution.
ASSAY
Procedure
Sample: 0.350 g of Lactobionic Acid
Analysis:  Dissolve the Sample in 50 mL of carbon dioxide-free water, previously heated to 30°. Immediately titrate with 0.1 N sodium hydroxide and determine the two equivalence points potentiometrically.
Each mL of 0.1 N sodium hydroxide consumed to the first equivalency point is equivalent to 35.83 mg of C12H22O12 (corresponds to the acid form), and each mL of 0.1 N sodium hydroxide consumed between the first and second equivalency points is equivalent to 34.03 mg of C12H20O11 (corresponds to the δ-lactone form).
Calculate the content, expressed as a percentage, of the lactobionic acid as the sum of both results.
Acceptance criteria: 98.0%–102.0% on the anhydrous basis

IMPURITIES
Water Determination
Acceptance criteria: 5.0% maximum.
Appearance of Solution
Sample solution: 120 mg/mL of Lactobionic Acid
Standard stock solution: Pipet 24.0 mL of ferric chloride CS and 6.0 mL of cobaltous chloride CS into a 100-mL volumetric flask. Dilute with 1% (w/v) hydrochloric acid to volume.
Reference solution:  Pipet 12.5 mL of the Standard stock solution into a 100-mL volumetric flask. Dilute with 1% (w/v) hydrochloric acid to volume.
Acceptance criteria: The Sample solution is clear and not more intensely colored than the Reference solution.
Optical Rotation
Sample solution: 10 mg/mL of Lactobionic Acid. Allow to stand for 24 h.
Acceptance criteria: +23.0° to +29.0° (anhydrous substance).
Reducing Sugars
Sample solution: Dissolve 5.0 g of Lactobionic Acid in 25 mL of water with the aid of gentle heat, and cool.
Analysis: To the Sample solution add 20 mL of cupric citrate TS and a few glass beads. Heat so that boiling begins after 4 min, and maintain boiling for 3 min. Cool rapidly, and add 100 mL of a 2.4% solution of glacial acetic acid and 20.0 mL of 0.025 M iodine VS. With continuous shaking, add 25 mL of a mixture of 6 mL of hydrochloric acid and 94 mL of water. When the precipitate has dissolved, titrate the excess iodine with 0.05 M sodium thiosulfate VS using 1 mL of starch TS, added toward the end of the titration as an indicator.
Acceptance criteria: NLT 12.8 mL of 0.05 M sodium thiosulfate is required, corresponding to NMT 0.2% of reducing sugars, as glucose.
Articles of Botanical Origin: 0.2% maximum.